GAO Caixia's team from the Institute of Genetics and Developmental Biology (IGDB) of the Chinese Academy of Sciences (CAS), together with collaborators, has recently developed a more efficient and improved plant prime editing system, Engineered Plant Prime Editor (ePPE).
Different from previously reported methods to improve the editing efficiency of prime editing through mainly optimizing the pegRNA, the researchers this time focused on the modification of the protein components in the prime editor. Related results were published on
Nature Biotechnology on March 24.
Prime editing is a newly described precision genome editing technology capable of generating precise base substitutions, deletions, or insertions of DNA bases at a specified target site in the genome of living cells.
In 2020, GAO's team successfully led the first establishment and optimization of using prime editing in rice and wheat through exploring the fusion of different reverse transcriptase enzymes, best experimental temperatures, pegRNA expression methods, primer binding site sequence and reverse transcriptase (RT) template sequence lengths and other conditions. Nevertheless, the editing efficiency of prime editors for use in plants was low and highly target dependent.
In this study, the researchers found that the deletion of the RNase H domain in the Moloney Murine Leukemia Virus (M-MLV) RT or fusion of a viral nucleocapsid protein at the N-terminus of the M-MLV RT greatly enhanced overall prime editing efficiencies in plant cells by 2.0-fold or 3.2-fold, respectively.
Furthermore, the researchers combined both approaches to develop the enhanced engineered plant prime editor, or ePPE. Compared with the original PPE, ePPE can increase editing efficiencies by an average of 5.8-fold when evaluating a variety of editing types, such as base substitutions, small fragment insertions, deletions, and insertions or deletions of larger fragments, without increasing the occurrence of off-target editing events or editing by-products.
When combining the newly developed ePPE with the dual-pegRNA strategy previously reported by GAO's team and the epegRNA (Engineered pegRNA) strategy developed by David Liu's group in 2021, the efficiency of the prime editing system for generating precise editing events at endogenous genes can be further improved.
Then the researchers used the ePPE system to successfully generate new rice plants that are resistant to two different herbicides, imazamox nicotinic acid and nicosulfuron.
In summary, the new ePPE system developed can drastically improve prime editing efficiencies in plants through engineering the protein components of PPE, and the combination of ePPE with pegRNA optimization strategies can further improve the editing efficiency across a variety of cell types.
This new editing system is expected to expand the utility of prime editing in agricultural breeding, crop improvement and more.
This research was supported by the National Natural Science Foundation of China and the Strategic Pilot Project A of CAS.
The establishment and application of ePPE, an efficient new genome editor for use in plants. a-b. Evaluating the original PPE with new prime editing variants using a fluorescent assay; c. Overall editing frequencies induced by PPE, PPE-ΔRNase H, PPE-NC-v1 and PPE-NC-v2. d, Schematic diagram of ePPE; e. Overall editing frequencies induced by PPE, PPE-ΔRNase H, PPE-NC-v1 and ePPE; f. Comparison of editing efficiencies of different PPEs with epegRNAs or dual-pegRNA strategy; g. Phenotype of an ALS-W548M rice mutant under different herbicide treatments. (Image by IGDB)
*GAO Caixia is a Doctoral supervisor of University of Chinese Academy of Sciences
Source: Chinese Academy of Sciences
Editor: GAO Yuan
